Hutchinson-Gilford progeria syndrome (HGPS) is the most dramatic human syndrome of premature aging. Children with this rare condition appear normal at birth, but by age 2 they have stopped growing, lost their hair, and shown skin changes and loss of subcutaneous tissue that resemble the ravages of old age. Untreated, they rarely live past adolescence, dying almost always of advanced cardiovascular disease (heart attack and stroke). Our laboratory discovered that 90% of cases of HGPS harbor a de novo point mutation in codon 608 of the LMNA gene. This mutation, denoted G608G, causes disease by creating an abnormal splice donor, generating an mRNA with an internal deletion of 150 nt. This is translated into a mutant form of the lamin A protein (referred to as progerin) that lacks 50 amino acids near the C-terminus. Normally lamin A is post-translationally processed to add a farnesyl group at the C-terminus, and then the last 18 amino acids are cleaved off by the enzyme ZMPSTE24 to produce mature lamin A. Progerin lacks the recognition site for this final cleavage, and so remains permanently farnesylated. We have shown that this abnormal protein acts as a dominant negative to disrupt the structure of the nuclear scaffold. Data from our group has also demonstrated that progerin interferes with proper chromosome segregation during mitosis, and alters the distribution of various histone chromatin marks. Our lab has developed a mouse model for HGPS, by inserting into the germline a 164 kb bacterial artificial chromosome (BAC) containing the human LMNA gene, engineered to carry the G608G mutation. Animals carrying the G608G mutation show progressive loss of smooth muscle cells in the media of large vessels. We tested the use of farnesyltransferase inhibitors (FTIs) in cell culture and the mouse model, to see if these drugs could provide benefit in HGPS by reducing the amount of progerin. The results were encouraging, and a four year open label clinical trial of FTIs in 30 children with the disease demonstrated benefit to the vascular system, with reduction in peripheral vascular resistance in most of the 28 children treated. This treatment is not a cure, however, and so the search for other therapeutic options continues. Homozygotes for the mouse G608G BAC transgenic have also now been bred, and show a considerably more severe phenotype, with death at 6-7 months of age. We have tested those animals to see if there might be therapeutic benefit from everolimus, a rapamycin analog, alone or in combination with FTIs. Rapamycin has been shown to extend lifespan in wild type mice, and its use on HGPS fibroblasts causes an improvement in phenotype with reduced nuclear blebbing and increased proliferative ability. We have shown that in cell culture, rapamycin acts by increasing turnover of progerin aggregates by activating autophagy. We conducted a drug trial in homozygous G608G mice to see if there might be therapeutic benefit from everolimus, and did see a modest improvement in lifespan. We have started a second trial on heterozygous G608G mice that are being treated with a combination of FTI and everolimus to determine if the drugs improve their vascular phenotype. We have also shown recently that everolimus has a beneficial effect on fibroblasts from patients with other LMNA mutations, including those that cause atypical Werners syndrome and Emery-Dreifuss muscular dystrophy. Based on the promising results from cell culture studies, and the encouraging safety profile of everolimus from long experience in organ transplantation, a Phase 1 trial of everolimus in children with progeria has just gotten underway at Boston Childrens Hospital. In yet another approach to the treatment of HGPS, we are investigating antisense oligonucleotide strategies to inhibit abnormal splicing at the cryptic G608G splice site. In collaboration with Sarepta Therapeutics, we tested several phosophorodiamidate morpholino oligonucleotides (PMOs) tiled across the cryptic G608G splice site in vitro in HGPS patient fibroblasts. Two of these candidate PMOs achieved reduction of progerin mRNA and protein, and were subsequently synthesized by Sarepta Therapeutics as proprietary peptide-conjugated phosophorodiamidate morpholino oligonucleotides (PPMOs). Using a GFP reporter system, we showed that intravenous and subcutaneous injection of PPMOs achieves excellent delivery to vascular smooth muscle cells of the mouse aorta. One of the PPMOs subsequently was shown to reduce progerin splicing in aorta, heart, and skeletal muscle in the G608G mouse model. Using this PPMO we have initiated a preclinical trial in our homozygous G608G transgenic mouse model. PPMO is being compared to saline control, administered by IV tail vein injection twice per week. Although mortality is due to myocardial infarction or stroke as a result of rapidly progressive atherosclerosis, HGPS patients also exhibit alopecia, bone and joint abnormalities, and subcutaneous fat loss. Thus cells and tissues derived from common mesenchymal progenitors are particularly susceptible to the pathology that results from the accumulation of progerin. Using a knock-in mouse model (LmnaG609G), we have initiated studies to characterize the molecular and cellular alterations in HGPS bone cell populations, which lead to defective tissue remodeling and progressive bone dysplasia. We are interested in knowing what would happen to normal cells or organisms if progerin production was completely prevented. To pursue this, we have created a mouse knock-in model that alters the sequence of mouse exon 11 to block the use of the cryptic splice site, without changing the encoded amino acid sequence. The knock-in heterozygotes and homozygotes are viable, and quantitative RNA measurements have confirmed that homozygotes make no progerin at all. Although progerin expression has been observed in human ductus arteriosus, and progerin is thought to play a role in the normal closure of this vessel, we did not observe any defects in ductus arteriosus closure in the homozygotes who make no progerin. Extensive histological analysis of the vessels and organs have thus far revealed no differences between the knock-in homozygotes and wild type mice. In a long term experiment, we are assessing whether these mice have exceptional longevity. Cell culture and whole animal studies of this progerin-free mouse strain are underway, and might reveal what role progerin plays in natural aging. Comprehensive understanding of how progerin drives HGPS phenotypes requires the identification of the protein interactors of lamin A/C and progerin. The high order insoluble structure of the nuclear envelope makes this challenging, even in cell culture, and has not been previously attempted in human tissues. We have developed a novel biotin proximity-based labeling approach and used it to identify lamin A/C and progerin interactors both in cell culture and directly from human tissue. We have identified instances where tissue culture did not faithfully replicate the interactome of primary tissues, and we have found multiple tissue-specific lamin A/C interactors. We have also identified proteins enriched or depleted in the presence of progerin. We have nowoptimized the method for quantitative comparative proteomics of primary human samples and are currently generating data and following up on initial findings.